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Krahmer, Natalie (2011): Analysis of Lipid Droplet Proteins and their Contribution to Phospholipid Homeostasis during Lipid Droplet Expansion. Dissertation, LMU München: Faculty of Biology
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Abstract

Lipid droplets (LDs) are storage organelles for neutral lipids. Recently, these organelles have been more and more recognized as dynamic structures with a complex and interesting biology. They store energy for later use and protect cells from lipotocixity caused by excess free fatty acids and cholesterol. Dysregulation of fat storage and mobilization, as well as excessive accumulation of LDs in tissues are key factors in pathogenesis of common diseases including obesity, insulin resistance, or hepatic steatosis. LDs have a unique physical structure. They are consisted of a neutral lipid core composed mainly of triglycerides (TG) and sterol esters (SE) that is surrounded by a phospholipid monolayer. Many proteins act on the LD surface to regulate LD functions. Despite considerable effort in determining the protein set of LDs, a reliable inventory of the LD proteome was so far missing. This thesis contains a first high confident LD proteome of Drosophila S2 cells that allows distinguishing between bona fide LD proteins and contaminants from other cellular organelles. Using a method called protein correlation profiling, I identified 106 proteins as candidates for LD proteins. Localization of a subset of these proteins by fluorescent microscopy confirmed LD targeting for more than 90% of the candidates. A comparison of proteomics data with genome-wide RNAi screens for genes whose knockdown alters LD morphology in S2 cells, revealed several LD proteins crucial for LD biology. One of them is CTP:phosphocholine cytidylyltransferase (CCT), the rate-limiting enzyme for phosphatidylcholine (PC) synthesis. Studying CCT targeting and function on the LD surface led me to the discovery of an elegant paradigm for the activation of PC synthesis by enzyme relocalization to maintain organelle phospholipid homeostasis. During conditions that promote lipid storage, LDs rapidly increase their core volume and surface area, and yet it was unknown how the need for surface phospholipids is sensed and balanced during this process. Here, I show that LDs require sufficient amount of PC, which acts as surfactant to prevent coalescence during their growth. PC synthesis for LD expansion is regulated by the activation of CCT, which binds to the surface of growing LDs. Activation of CCT by LD targeting is reversible and correlates with the need for PC at LDs, and thus may be part of a homeostatic feedback loop regulating PC synthesis. The localization and requirements for CCT were similar in Drosophila and murine cell lines, indicating evolutionary conservation.